Bacteria in the periodontal pocket can develop complex sessile communities that play a significant role in infection-induced periodontal disease. Data emerging from the oral microbiome project is facilitating a shift in the paradigm for infection-induced periodontal diseases. Bacteria like Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter (Actinobacillus) actinomycetemcomitans, Tannerella forsythia and Treponema denticola have previously been demonstrated to be major pathogens associated with periodontal diseases. However, recent developments including novel, culture-independent techniques have allowed the identification of as-yet-culturable and fastidious organisms in patients suffering from periodontitis. Collectively, these studies have demonstrated that changes in periodontal status are associated with shifts in the composition of the bacterial community in the periodontal pocket. Filifactor alocis, a Gram-positive, asaccharolytic, obligate anaerobic rod, is one of the marker organisms that is now identified to be significant to the pathogenetic structure of biofilms associated with periodontal inflammation and should be considered an important organism for periodontal disease. Further, in comparison to the other traditional periodontal pathogens, F. alocis is present in the periodontal pocket in higher numbers and is least detected in healthy or periodontitis resistant patients. Currently, there is little or no information on the pathogenesis of F. alocis. In our laboratory, preliminary studies of F. alocis showed non-gingipain protease and sialidase activities. In silico analysis revealed molecular relatedness of several virulence factors from F. alocis and P. gingivalis. F. alocis was more resistant to oxidative stress and its growth stimulated under those condition in contrast to P. gingivalis. Biofilm formation was significantly increased in co-culture. There was an increase in adherence and invasion of epithelial cells in co-culture compared with P. gingivalis or F. alocis mono-cultures. In those epithelial cells, endocytic vesicle mediated internalization was only observed during co-culture. The F. alocis clinical isolate D-62D had an increased invasive capacity in co-culture with P. gingivalis compared to the F. alocis ATCC 35896 strain. In addition, there was variation in the proteome of the clinical isolates compared to the ATCC 35896 strain. Hypothetical proteins and those known to be important virulence factors in other bacteria were indentified. Our observations, taken together, may suggest that F. alocis has virulence properties that may enhance its ability to survive/persist in the periodontal pocket and may play an important role in infection-induced periodontal disease. It is our hypothesis that in F. alocis, similar to other periodontal pathogens, multiple coordinately regulated mechanisms are significant in the pathogenicity of the organism. We wish to gain an understanding of F. alocis potential virulence factors and evaluate whether they contribute to its pathogenicity. The primary goal of this R21 application is to develop a genetic system and an animal model to evaluate the host response to and virulence potential of F. alocis. The specific aims are: (1) To evaluate the specific role(s) of protease activity in the survival/pathogenicity of F. alocis. (2) o develop an animal model for testing the virulence potential of F. alocis. Collectively, the results from this study will establish basic information on the pathogenesis of F. alocis. It will generatea model system(s) that will yield important clues that will facilitate the development of novel therapeutic interventions to aid in the control and prevention of periodontal disease